The circadian clock, or circadian oscillator, in most living things makes it possible for organisms to coordinate their biology and behavior with daily and seasonal changes in the day-night cycle. The clock is a biochemical mechanism that oscillates with a period of exactly 24 hours when it receives daily corrective signals from the environment, primarily daylight and darkness. Circadian clocks are the central mechanisms which drive circadian rhythms. They consist of three major components:
- A central oscillator with a period of about 24 hours that keeps time
- A series of input pathways to this central oscillator to allow entrainment of the clock
- A series of output pathways tied to distinct phases of the oscillator that regulate overt rhythms in biochemistry, physiology, and behavior throughout an organism
The clock is reset as the environment changes through an organism's ability to sense external time cues of which the primary one is light. Circadian oscillators are ubiquitous in tissues of the body where they are synchronized by both endogenous and external signals to regulate transcriptional activity throughout the day in a tissue-specific manner. The circadian clock is intertwined with most cellular metabolic processes and it is affected by organism aging. The basic molecular mechanisms of the biological clock have been defined in vertebrate species, Drosophila melanogaster, plants, fungi, bacteria, and presumably also in Archaea.
Transcriptional and translational control
Evidence for a genetic basis of circadian rhythms in higher eukaryotes began with the discovery of the period (per) locus in Drosophila melanogaster from forward genetic screens completed by Ron Konopka and Seymour Benzer in 1971. Through the analysis of per circadian mutants and additional mutations on Drosophila clock genes, it was demonstrated that there is an underlying generative molecular mechanism of the circadian clock that consists of a set of core clock genes and their protein products, which together participate in positive and negative autoregulatory feedback loops of transcription and translation. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Similar mechanisms have been demonstrated in mammals and other organisms.
Several mammalian clock genes have been identified and characterized through experiments on animals harboring naturally occurring, chemically induced, and targeted knockout mutations, and various comparative genomic approaches. The majority of identified clock components are transcriptional activators or repressors that modulate protein stability and nuclear translocation, and create two interlocking feedback loops. In the primary feedback loop, members of the basic helix-loop-helix (bHLH)-PAS (Period-Arnt-Single-minded) transcription factor family, CLOCK and BMAL1, heterodimerize in the cytoplasm to form a complex that, following translocation to the nucleus, initiates transcription of target genes such as the core clock genes Period genes (PER1, PER2, and PER3) and two Cryptochrome genes (CRY1 and CRY2). Negative feedback is achieved by PER:CRY heterodimers that translocate back to the nucleus to repress their own transcription by inhibiting the activity of the CLOCK:BMAL1 complexes. Another regulatory loop is induced when CLOCK:BMAL1 heterodimers activate the transcription of Rev-ErbA and Rora, two retinoic acid-related orphan nuclear receptors. REV-ERBa and RORa subsequently compete to bind retinoic acid-related orphan receptor response elements (ROREs) present in Bmal1 promoter. Through the subsequent binding of ROREs, members of ROR and REV-ERB are able to regulate Bmal1. While RORs activate transcription of Bmal1, REV-ERBs repress the same transcription process. Hence, the circadian oscillation of Bmal1 is both positively and negatively regulated by RORs and REV-ERBs.
In D. melanogaster, the gene cycle (CYC) is the orthologue of BMAL1 in mammals. Thus, CLOCK–CYC dimers activate the transcription of circadian genes. The gene timeless (TIM) is the orthologue for mammalian CRYs as the inhibitor; D. melanogaster CRY functions as a photoreceptor instead. In flies, CLK–CYC binds to the promoters of circadian-regulated genes only at the time of transcription. A stabilizing loop also exists where the gene vrille (VRI) inhibits whereas PAR-domain protein-1 (PDP1) activates Clock transcription. In N. crassa, the clock mechanism is analogous, but non-orthologous, to that of mammals and flies.
For a long time it was thought the transcriptional activation/repression cycles driven by the transcriptional regulators constituting the circadian clock was the main driving force for circadian gene expression in mammals. More recently, however, it was reported that only 22% of messenger RNA cycling genes are driven by de novo transcription. RNA-level post-transcriptional mechanisms driving rhythmic protein expression were later reported, such as mRNA polyadenylation dynamics.
Fustin and co-workers identified methylation of internal adenosines (m6A) within mRNA (notably of clock transcripts themselves) as a key regulator of the circadian period. Inhibition of m6A methylation via pharmacological inhibition of cellular methylations or more specifically by siRNA-mediated silencing of the m6A methylase Mettl3 led to the dramatic elongation of the circadian period. In contrast, overexpression of Mettl3 in vitro led to a shorter period. These observations clearly demonstrated the importance of RNA-level post-transcriptional regulation of the circadian clock, and concurrently established the physiological role of (m6A) RNA methylation.
The autoregulatory feedback loops in clocks take about 24 hour to complete a cycle and constitute a circadian molecular clock. This generation of the ~24-hour molecular clock is governed by post-translational modifications such as phosphorylation, sumoylation, histone acetylation and methylation, and ubiquitination. Reversible phosphorylation regulates important processes such as nuclear entry, formation of protein complexes and protein degradation. Each of these processes significantly contributes to keeping the period at ~24 hours and lends the precision of a circadian clock by affecting the stability of aforementioned core clock proteins. Thus, while transcriptional regulation generates rhythmic RNA levels, regulated posttranslational modifications control protein abundance, subcellular localization, and repressor activity of PER and CRY.
Proteins responsible for post-translational modification of clock genes include casein kinase family members (CSNK1D and CSNK1E) and the F-box and leucine-rich repeat protein 3 (FBXL3). In mammals, Casein kinase 1 epsilon and Casein kinase 1 delta are critical factors that regulate the core circadian protein turnover. Experimental manipulation on either of these proteins results in dramatic effects on circadian periods, such as altered kinase activities and cause shorter circadian periods, and further demonstrates the importance of the post-translational regulation within the core mechanism of the circadian clock. These mutations have become of particular interest in humans as they are implicated in familial advanced sleep phase syndrome. A small ubiquitin-related modifier protein modification of BMAL1 has also been proposed as another level of post-translational regulation.
Regulation of circadian oscillators
Circadian oscillators are simply oscillators with a period of approximately 24 hours. In response to light stimulus the body corresponds with a system and network of pathways that work together to determine the biological day and night. The regulatory networks involved in keeping the clock precise span over a range of post-translation regulation mechanisms. Circadian oscillators may be regulated by phosphorylation, SUMOylation, ubiquitination, and histone acetylation and deacetylation, the covalent modification of the histone tail which controls the level of chromatin structures causing the gene to be expressed more readily. Methylation of a protein structure adds a methyl group and regulates the protein function or gene expression and in histone methylation gene expression is either suppressed or activated through changing the DNA sequence. Histones go through an acetylation, methylation and phosphorylation process but the major structural and chemical changes happen when enzymes histone acetyltransferases (HAT) and histone deacetylases (HDAC) add or remove acetyl groups from the histone causing a major change in DNA expression. By changing DNA expression, histone acetylation and methylation regulate how the circadian oscillator operates. Fustin and co-workers provided a new layer of complexity to the regulation of circadian oscillator in mammals by showing that RNA methylation was necessary for efficient export of mature mRNA out of the nucleus: inhibition of RNA methylation caused nuclear retention of clock gene transcripts, leading to a longer circadian period.
Systems biology approaches to elucidate oscillating mechanisms
Modern experimental approaches using systems biology have identified many novel components in biological clocks that suggest an integrative view on how organisms maintain circadian oscillation.
Recently, Baggs et al. developed a novel strategy termed "Gene Dosage Network Analysis" (GDNA) to describe network features in the human circadian clock that contribute to an organism's robustness against genetic perturbations. In their study, the authors used small interfering RNA (siRNA) to induce dose-dependent changes in gene expression of clock components within immortalized human osteosarcoma U2OS cells in order to build gene association networks consistent with known biochemical constraints in the mammalian circadian clock. Employing multiple doses of siRNA powered their quantitative PCR to uncover several network features of the circadian clock, including proportional responses of gene expression, signal propagation through interacting modules, and compensation through gene expression changes.
Proportional responses in downstream gene expression following siRNA-induced perturbation revealed levels of expression that were actively altered with respect to the gene being knocked down. For example, when Bmal1 was knocked down in a dose-dependent manner, Rev-ErbA alpha and Rev-ErbA beta mRNA levels were shown to decrease in a linear, proportional manner. This supported previous findings that Bmal1 directly activates Rev-erb genes and further suggests Bmal1 as a strong contributor to Rev-erb expression.
In addition, the GDNA method provided a framework to study biological relay mechanisms in circadian networks through which modules communicate changes in gene expression. The authors observed signal propagation through interactions between activators and repressors, and uncovered unidirectional paralog compensation among several clock gene repressors—for example, when PER1 is depleted, there is an increase in Rev-erbs, which in turn propagates a signal to decrease expression in BMAL1, the target of the Rev-erb repressors.
By examining knockdown of several transcriptional repressors, GDNA also revealed paralog compensation where gene paralogs were upregulated through an active mechanism by which gene function is replaced following knockdown in a nonredunant manner—that is, one component is sufficient to sustain function. These results further suggested that a clock network utilizes active compensatory mechanisms rather than simple redundancy to confer robustness and maintain function. In essence, the authors proposed that the observed network features act in concert as a genetic buffering system to maintain clock function in the face of genetic and environmental perturbation. Following this logic, we may use genomics to explore network features in the circadian oscillator.
Another study conducted by Zhang et al. also employed a genome-wide small interfering RNA screen in U2OS cell line to identify additional clock genes and modifiers using luciferase reporter gene expression. Knockdown of nearly 1000 genes reduced rhythm amplitude. The authors found and confirmed hundreds of potent effects on period length or increased amplitude in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling pathway, the cell cycle, and folate metabolism. Coupled with data demonstrating that many of these pathways are clock-regulated, Zhang et al. postulated that the clock is interconnected with many aspects of cellular function.
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